The long-range goals of our studies are to understand the mechanisms that enforce the ??T and pre-TCR checkpoints. We would like to describe these checkpoints in terms of global networks involving transcriptional regulators, signaling components and survival factors. Previously, we as well as others have demonstrated that E- and Id-proteins play critical roles in enforcing the ??, pre-TCR and TCR checkpoints. Most prominent among the E-proteins are the E2A gene products, E12 and E47. Two additional members that are closely related to E2A, named E2-2 and HEB, also belong to the E-protein family. The DNA binding activities of E-proteins are attenuated by a subset of helix-loop-helix (HLH) proteins, named ld1-4. E-proteins levels are high in T cell progenitors where they initiate TCRy, ? as well ? locus rearrangement, activate the expression of genes encoding for proteins involved in Notch- and pre-TCR signaling arid antagonize proliferation. Once a ?? or pre-TCR complex is assembled, IdS levels are elevated to suppress E2A DNA binding. Here we propose to continue these studies. We would use functional studies, genome-wide analyses and computational approaches to determine the mechanisms that underpin ?? versus ? selection. We would identify factors that cooperate with E-proteins to enforce the pre-TCR checkpoints. We would examine whether gradients of E-protein activity modulate DNA binding site preferences as well as enhancer repertoire selection beyond the pre-TCR checkpoint. We would examine how Notch- and pre-TCR signaling act in concert to modulate binding site as well as enhancer selection. We would describe ?? T cell development in terms of global networks of enhancer repertoires, interacting transcription factors, signaling components and survival factors. We would examine how differences in signaling by the pre- TCR and ?? TCR affect E2A occupancy and binding site selection. Finally, we would examine how these networks change during developmental progression and how such changes relate to enforcement of the ?? and pre-TCR checkpoints. RELEVANCE (See instructions): It has been established that an important population of cells, named ?? T cells, play critical roles in preserving epithelial structures that function as barriers. The proposal described here is aimed to understand the molecular mechanisms that promote their developmental progression. These studies may permit novel avenues for the treatment of immune diseases and interference with the development of malignancies.